Abstract

Activation of group 1 metabotropic glutamate receptors (mGluRs) induces long-term depression (LTD) of synaptic transmission that relies on dendritic protein synthesis. We investigated the signal transduction pathways required for mGluR-LTD to identify candidate mechanisms for mGluR regulation of synaptic protein synthesis. Our results demonstrate a role for extracellular signal-regulated protein kinase (ERK), a subclass of the mitogen-activated protein kinases (MAPKs), in mGluR-LTD in area CA1 of the rat hippocampus. Inhibitors of the upstream kinase of ERK, MAP/ERK kinase significantly reduce mGluR-LTD induced by the group 1 agonist dihydroxyphenylglycine (DHPG) and synaptic stimulation but do not affect NMDA receptor-dependent LTD. In contrast, inhibitors of p38 MAPK were ineffective against DHPG-induced LTD. Consistent with the role of ERK in mGluR-LTD, we observed that DHPG treatment of hippocampal slices (isolated CA1), at concentrations that induce LTD, results in a robust phosphorylation of ERK but not of p38 MAPK. These results point to ERK as an important regulator of mGluR-LTD and a potential mechanism for mGluR regulation of synaptic protein synthesis.

Here we investigated the signal transduction pathways specifically required for protein synthesis-dependent LTD induced by DHPG and PP-LFS in area CA1 of juvenile rats. Surprisingly, our experiments reveal a role for ERK rather than p38 MAPK in this form of LTD. These results demonstrate a role for ERK in group 1 mGluR-mediated synaptic depression and implicate the ERK pathway as a candidate mechanism for the regulation of synaptic protein synthesis by mGluRs.

p38 MAPK is not required for DHPG-induced LTD in area CA1. A, B, Application of DHPG (100 μm, 5 min, in 0.1% DMSO) to hippocampal slices induced LTD in FPs recorded in the dentate gyrus (A) and area CA1 (C). B, D, Preapplication of the p38 MAPK inhibitor SB203580 (5 μm, 1 hr) prevents LTD in the dentate gyrus (B) but does not affect LTD in area CA1 (D). Dashed lines indicate 100% of baseline.

DHPG at concentrations that induce LTD and activate the ERK cascade, but not p38 MAPK, in CA1. Representative Western blots are shown of untreated [ACSF or basal (B)] or treated [100 μm DHPG (D), 5 min] hippocampal slices. Quantitative group data of each condition are described below. The number of slices (n) is indicated on each bar. Conditions are indicated below each bar (D 50 and D 100 indicate 50 and 100 μm DHPG, respectively, for 5 min). *p < 0.01, statistically different from condition control. A, DHPG-induced ERK phosphorylation does not rely on NMDAR activation or action potentials and is blocked by U0126. B, DHPG-induced ERK phosphorylation is blocked by the group 1 mGluR antagonists LY367385 (LY) and MPEP. C, DHPG increases the phosphorylation of the ERK substrate RSK1 and is blocked by U0126. Representative Western blots show control (C) extract (phorbol ester-treated HeLa cells) and hippocampal slices (basal) or DHPG treated as indicated. D, In contrast, DHPG treatment does not result in an increase in phosphorylated p38 MAPK/total p38 MAPK. Dashed lines indicate 100% of basal.

To determine whether DHPG induces phosphorylation of p38 MAPK, we performed Western blots on hippocampal slices with phosphospecific (Thr180/Tyr182) antibodies to p38 MAPK. Unlike ERK, we were not able to detect significant increases in p38 MAPK phosphorylation after DHPG treatment (100 μm, 5 min, 112 ± 7%; n = 7; p = 0.12). Together, our results suggest that there are differences in the signal transduction mechanisms required for mGluR-LTD in the dentate gyrus and area CA1.

Discussion

Our data demonstrate a requirement for ERK in mGluR-LTD and suggest that ERK may also be an important signaling molecule for regulation of protein synthesis at synapses. Surprisingly, we did not find a role for p38 MAPK in DHPG-induced LTD. Consistent with the involvement of ERK in LTD, we observed a robust phosphorylation of ERK and RSK in response to DHPG at concentrations that induce LTD in hippocampal brain slices. These findings demonstrate a functional role for ERK in mGluR-dependent synaptic depression in the hippocampus and provide candidate signaling pathways for mGluR regulation of synaptic protein synthesis.

Role of ERK in mGluR-dependent synaptic plasticity

It has been demonstrated previously that the group 1 mGluR agonist DHPG activates ERK in hippocampal slices (Roberson et al., 1999). However, a role for ERK in mGluR-mediated physiological changes has only been described recently (Coogan et al., 1999). Here, we have shown a selective effect of MEK inhibitors on LTD induced with DHPG and synaptic stimulation. In contrast, NMDAR-dependent LTD induced with LFS is unaffected (Fig. 1). Consistent with our finding, a recent study in slice culture found that the MEK inhibitor PD98059 did not interfere with NMDAR-dependent LTD (Zhu et al., 2002). Because DHPG-induced and PP-LFS-induced LTD are both dependent on dendritic protein synthesis, as opposed to NMDAR-dependent LTD, these results suggest that ERK may be important in regulating synaptic protein synthesis. However, the effects of the MEK inhibitors were not as rapid or robust as the effects of protein synthesis inhibitors, especially in the case of PP-LFS (Huber et al., 2000). The incomplete blockade of mGluR-LTD by U0126 is not attributable to an incomplete blockade of ERK activation (Fig. 3A). Therefore, this suggests that other signaling pathways may play an earlier or additional role in mGluR-LTD.

There is previous evidence for ERK in hippocampal LTD in vivo. A previous study in area CA1 in vivo found that systemic administration of the MEK inhibitorα-[amino[(4-aminophenyl) thio]methylene]-2-(trifluoromethyl)benzeneacetonitrile (SL327) inhibits LTD (Thiels et al., 2002). Although the induction of LTD is this study required NMDARs (Thiels et al., 1994; Heynen et al., 1996), there is evidence for a later mGluR-dependent and protein synthesis-dependent phase in vivo (Manahan-Vaughan, 1997; Manahan-Vaughan et al., 2000). Indeed, ERK and protein synthesis are also required for mGluR-LTD at granule cell to Purkinje neuron synapses (Ahn et al., 1999; Kawasaki et al., 1999; Karachot et al., 2001). Together, these data suggest that ERK is important in long-term protein synthesis-dependent synaptic plasticity in vivo, in vitro, and across brain regions.

We observed that DHPG activated ERK and its downstream effector, RSK1. Although RSK1 has been traditionally studied for its role in transcriptional regulation, there is some evidence for its role in protein synthesis regulation in the hippocampus. MGluR activation of hippocampal slices induces the translocation of RSK1 to polyribosomes and a concomitant increase in the phosphorylation of selective polyribosome proteins thought to be mediated by RSK1 (Angenstein et al., 1998). There is also emerging evidence for ERK and RSK in translation regulation in nonneuronal cells (Herbert et al., 2000; Wang and Proud, 2002). Future studies are aimed at determining whether DHPG activates these regulatory pathways in hippocampal neurons.

Differential role of p38 MAPK in mGluR-LTD

There is growing evidence for a role of p38 MAPK in LTD in a number of contexts (Bolshakov et al., 2000; Rush et al., 2002; Zhu et al., 2002). Although we confirmed the role of p38 MAPK in mGluR-LTD in the dentate gyrus, p38 MAPK inhibitors do not inhibit mGluR-LTD in adolescent CA1. Consistent with these data, DHPG does not activate p38 MAPK in area CA1. Our slices contained dentate gyrus, so it is surprising that we were not able to detect p38 MAPK activation the slices. However, this could be caused by a signal-to-noise problem, and a comparison of DHPG-stimulated p38 MAPK in the dentate gyrus versus CA1 subregions may be required. Our data suggest that p38 MAPK may be differentially required for LTD depending on the brain region.

Role for ERK in long-term potentiation and LTD?

Our data demonstrating a role for ERK in mGluR-dependent LTD add to the growing body of literature implicating ERK in long-term synaptic plasticity (for review, see Sweatt, 2001). The most prominent role for ERK has been in the induction of long-term potentiation (LTP) through activation of NMDARs or mGluRs (Coogan et al., 1999; Sweatt, 2001). However, our data and those of Thiels et al. (2002) implicate ERK in LTD. In particular, we propose that ERK is specifically important in mGluR- and protein synthesis-dependent LTD. Very recent data indicate that ERK is an important regulator of the protein synthesis-dependent phase of LTP and regulates neuronal protein synthesis in response to neuronal activity and BDNF (Kelleher et al., 2004). How can ERK signal to induce both protein synthesis-dependent LTP and LTD?

One hypothesis to consider is that ERK is a general regulator of neuronal protein synthesis in response to different extracellular stimuli, and the specificity of the response (LTP or LTD) may be determined by the pattern of synaptic activity. Alternatively, the activation of ERK may be differentially regulated by NMDARs and mGluRs via different small GTPases. Recent work suggests that Rap1 regulates a specific subcellular fraction of ERK activity in hippocampal neurons (Morozov et al., 2003). It will be very interesting to learn how ERK and protein synthesis-dependent mechanisms lead to either LTP or LTD.

Footnotes

This research was supported in part by the National Institutes of Health, Howard Hughes Medical Institute (M.F.B.), and McKnight and FRAXA Research Foundations (K.M.H.). K.M.H. is a Southwestern Medical Foundation endowed scholar in biomedical research. We thank Gray Pearson and Melanie Cobb for helpful discussions and technical assistance with the Western blots.

Zho WM, You JL, Huang CC, Hsu KS (2002) The group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine induces a novel form of depotentiation in the CA1 region of the hippocampus. J Neurosci22: 8838–8849.